DNA transposons are discrete DNA sequences that can move (transpose) from one location in the genome to another. They are present in virtually all organisms and contribute to both genome structure and function. DNA transposons are also natural gene delivery vehicles that are being developed as tools for genome engineering, such as insertional mutagenesis and transgenesis, and for human gene therapy. The reaction of DNA transposition occurs within a protein-DNA complex (transpososome), which contains two or more transposase enzymes and the ends of the transposon DNA. Despite the significant progress in understanding the DNA transposition has been made using biochemical and molecular genetics approaches, fundamental questions about the functional and structural mechanisms underpinning transpososome assembly and operation remain unaddressed. The long-term objective of my laboratory is to elucidate these mechanisms and how they can be exploited for different genetic applications. In this project we propose to investigate a Sleeping Beauty (SB) DNA transposon, the most widely used transposon in research genetic applications and the first and only DNA transposon in clinical trials for human gene therapy. The specific objective of this proposal is to obtain the critically needed high-resolution structural and dynamics information on SB transposase and its binding to DNA. Our experimental approach integrates advanced solution NMR techniques, mutagenesis, and biochemical functional assays. The specific aims of this proposal are directed at (1) identifying key functionally important structural features of the SB transposase, (2) the functional dynamics of the SB transposase, and (3) establishing the role of the RED subdomain of SB transposase in the transposon DNA recognition. The results will have broad applications to understanding the functional structural and dynamics features of DNA transposases and significant particular applications to human gene therapy and gene delivery to animal cells using the SB transposon.